Current sequencing projects
Neurogenetics laboratorySomatic mutation detection (Frank Baas, Marja Jakobs)
Laboratory of Experimental VirologyVirus discovery (Michel de Vries)
Dept. Clinical Immunology & RheumatologyTCR-beta variant detection (Niek de Vries, Paul Klarenbeek)
Clinical VirologyHepatitis C (Richard Molenkamp)
Erasmus MC – RotterdamRe-sequencing tumor samples (Ernie de Boer, Michael Moorhouse)
TCR-beta
Recombinatie van gensegmenten :
Vanuit de germline:
- Van ieder gensegment wordt 1 variant geselecteerd
-Deze worden aan elkaar gekoppeld
Alleen functionele recombinaties leiden tot functionele T-cellen
Paul Klarenbeek
Totale theoretische variatie(in vivo blijkt dit veel lager te liggen)
Paul Klarenbeek
CDR3 region
Unique for each clonal expansion
How to identify clonal expansions?
Germline DNA
mRNA
Thymocytes
Paul Klarenbeek
Dept. Clinical Immunology& Rheumatology
Goal: identify and enumerate TCR-beta variants
Vn CDR3 JPrimer A
J
C
Primer B
Barcode
Vn CDR3 JPrimer A
C
C
Primer B
Barcode
Paul Klarenbeek
Roche (454) sequencing
Run 07-07-2008110,509 sequences
26,445,844 nucleotides in total
Run 30-09-2008106,234 sequences
24,983,646 nt
Sequence lengthsFrequency sequence length
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
0 50 100 150 200 250 300 350 400 450
sequence length
freq
uen
cy
frequency sequence length
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
0 50 100 150 200 250 300
sequence length
freq
uen
cy
20080707
20080930
Pipeline Rheumatology
Convert sff to fasta+quality scores
Chop sequences into: MID, fragment
Divide sequences based on MID and region
Identify the V, J and C segments
Locate highly variable area
Count variants Quality control
Perl scriptsRoche softwareBLATAccess/Excel
Recognize MID and primer
MID = barcode for samplePrimer = Primer for J or C segment
# MIDs for the 3 regions that are sequenced with primer for J segment:
MID=(TAGT|ACTA|CGAC|CTCG)# Primer for J segment (first) or C segment (last two):Primer=(CTTACCTACAACGGTTAACCTGGTC|
AGCTCAAACACAGCGACCTC|GGAACACCTTGTTCAGGTC)
Pipeline Rheumatology
Convert sff to fasta+quality scores
Chop sequences into: MID, fragment
Divide sequences based on MID and region
Identify the V, J and C segments
Locate highly variable area
Count variants Quality control
Perl scriptsRoche softwareBLATAccess/Excel
Identify V, J and segment
IMGT website: reference sequencesBLAT all roche sequences against 3
referencesSelection: only store first hit per reference
MID C J CDR3 V
MID J CDR3 V
Locate highly variable region(CDR3)
Get CDR3 sequence -> countDetermine deletions from V and J segment
Determine reading frame
MID C J CDR3 V
MID J CDR3 V
Pipeline Rheumatology
Convert sff to fasta+quality scores
Chop sequences into: MID, fragment
Divide sequences based on MID and region
Identify the V, J and C segments
Locate highly variable area
Count variants Quality control
Perl scriptsRoche softwareBLATAccess/Excel
Count variants: BLAT hits
Blat hits TRBV reference
0
1000
2000
3000
4000
5000
6000X
0719
2|T
RB
V12
-3*0
1|H
omo
X07
223|
TR
BV
12-5
*01|
Hom
o
X07
192|
TR
BV
12-3
*01|
Hom
o
X07
223|
TR
BV
12-5
*01|
Hom
o
M14
264|
TR
BV
12-4
*02|
Hom
o
X07
192|
TR
BV
12-3
*01|
Hom
o
X07
223|
TR
BV
12-5
*01|
Hom
o
X07
192|
TR
BV
12-3
*01|
Hom
o
X07
223|
TR
BV
12-5
*01|
Hom
o
X07
223|
TR
BV
12-5
*01|
Hom
o
X07
192|
TR
BV
12-3
*01|
Hom
o
X07
223|
TR
BV
12-5
*01|
Hom
o
X07
192|
TR
BV
12-3
*01|
Hom
o
X74
844|
TR
BV
7-9*
06|H
omo
X07
192|
TR
BV
12-3
*01|
Hom
o
X07
223|
TR
BV
12-5
*01|
Hom
o
X07
192|
TR
BV
12-3
*01|
Hom
o
X07
223|
TR
BV
12-5
*01|
Hom
o
M14
264|
TR
BV
12-4
*02|
Hom
o
ACTA ACTA ACTA ACTA ACTA CGAC CGAC CTCG CTCG nomatch nomatch nomatch nomatch nomatch TAGT TAGT TAGT TAGT TAGT
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
region/MID/hit
freq
uen
cy
Count CDR3 variantsregion cdr3_sequence freq
1 767
1 CTCCCTTTTTGGGGG 32
1 CCCTCAGGATTTCAGGG 30
1 TGGGTC 29
1 CAAACATGA 23
1 GGGACGGAGA 20
1 GGACAGT 20
1 CCCAGACAGG 19
1 AACCGGA 18
1 CTCCACTGGACACGT 18
1 ACGGG 18
1 CGCCCGGGACAGGGCCCTTCGGGG 18
1 CCACCCCGCGGCAGGAGGG 17
1 CCCACCGGGACAGGGGCGTC 17
1 GGTATACGGGCAGCGG 16
1 GACCTTGTGGTC 16
1 ACAGGGGGAG 16
1 GCGGG 16
Example of CDR3
sequences
V segment deletions
0
5000
10000
15000
20000
25000
30000
35000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 19 21 22 23 24 25 26 28 29 31 32
nt
fre
qu
en
cy
Count deleted ntof V and J segment
Check where alignment stops wrt reference
J segment deletions
0
5000
10000
15000
20000
25000
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
nt
freq
uen
cy
Pipeline Rheumatology
Convert sff to fasta+quality scores
Chop sequences into: MID, fragment
Divide sequences based on MID and region
Identify the V, J and C segments
Locate highly variable area
Count variants Quality control
Perl scriptsRoche softwareBLATAccess/Excel
To do next
Determine reading frameQuality control
Future plansDetection of all TCR-beta variants
TCR-alpha receptor variantsSame procedure for B-cells
Screen patients for receptor variations
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